We would like to thank Dr. Bryan E. Tomlin affiliated with the elemental analysis lab within the department of chemistry at TAMU who assisted with the neutron activation analysis (NAA).

1. Polycaprolacyone triacrylate (PCLTA) synthesis
<ol>
	<li>Dry 3.2 mL of 400 Da polycaprolacyone (PCL) triol overnight at 50 &#176;C in an open 200 mL round bottom flask and K2CO3 in a glass vial at 90 &#176;C.</li>
	<li>Mix the triol with 6.4 mL of dichloromethane (DCM) and 4.246 g of potassium carbonate (K2CO3) under argon.</li>
	<li>Mix 2.72 mL of acryloyl chloride in 27.2 mL of DCM and add dropwise to the reaction mixture in the flask over 5 min.</li>
	<li>Cover the reaction mixture with aluminum foil and leave it undisturbed at room temperature for 24 h under argon.</li>
	<li>After 24 h, filter the reaction mixture using a filter paper on a Buchner funnel under vacuum to discard excess reagents.</li>
	<li>Precipitate filtrate from step 1.5 that contains the endcapped polymer in diethyl ether in a 1:3 (vol/vol) and rotovape at 30 &#176;C to remove the diethyl ether.</li>
</ol>
2. Formation of the polybubble
NOTE: Injecting polymer in the deionized (DI) water would cause the polybubbles to migrate to the bottom of the vial resulting in flattened bottom. Use 10% (wt/vol) carboxymethyl cellulose (CMC) fill the glass vial instead to avoid polybubble flattening.
<ol>
	<li>Prepare 10% (wt/vol) CMC solution in DI water.</li>
	<li>Fill a 0.92 mL glass vial with 0.8 mL of 10% CMC using a 1 mL transfer pipet.</li>
	<li>Mix 1000 mg/mL of 14 kDa PCL in DCM and synthesize PCLTA in a 1:3 (vol/vol) for a total volume of 200 &#181;L or prepare 200 &#181;L of 1000 mg/mL of 5 kDa poly (lactic-co-glycolic acid) diacrylate (PLGADA) in chloroform.</li>
	<li>Mix the 2-hydroxy-4&#8242;-(2-hydroxyethoxy)-2-methylpropiophenone (photoinitiator) with the polymer (PLGADA or PCL/PCLTA) mixture in 0.005:1 (vol/vol).</li>
	<li>Load 200 &#181;L of polymer mixture into a 1 mL glass syringe mounted on a syringe pump that is connected to a dispensing stainless-steel tube with an inner diameter of 0.016 inch.</li>
	<li>Use a micromotor to control the forward and backward motion of the polymer tube to inject polymer into the 10% CMC in the glass vial to form the polybubble.</li>
	<li>Cure the polybubbles under ultraviolet (UV) at 254 nm wavelength for 60 s at 2 W/cm2.</li>
	<li>Flash freeze the polybubbles in liquid nitrogen and lyophilize overnight at 0.010 mBar vacuum and at -85 &#176;C.</li>
	<li>Separate the polybubbles from the dried CMC using forceps and wash the polybubbles with DI water to remove any residual CMC. Note that other polymers can be used likely with modifications to alter the release kinetics.</li>
</ol>
3. Modulation of polybubble diameter
<ol>
	<li>Fill a 0.92 mL glass vial with 10% CMC using a 1 mL transfer pipet.</li>
	<li>Mix PCL/PCLTA in a 1:3 (vol/vol) with 1000mg/mL 14kDa PCL and synthesize PCLTA. Mix the photoinitiator with polymer mixture in a 0.005:1 (vol/vol).</li>
	<li>Load the polymer mixture into a 1 mL glass syringe mounted on a syringe pump that is connected to a dispensing stainless-steel tube with an inner diameter of 0.016 inch.</li>
	<li>Use a micromotor to control the forward and backward motion of the polymer tube to inject polymer into the 10% CMC in the glass vial to form the polybubble.</li>
	<li>To obtain polybubbles with various diameters, vary dispensing rate from 0.0005 to 1 &#181;L/s.</li>
	<li>Take images of the vial with the polybubbles with varying diameter.</li>
	<li>Use ImageJ to quantify the diameter of the polybubbles and use the size of the vial as scale.</li>
</ol>
4. Centering cargo within polybubble
<ol>
	<li>Modulation of PCL/PCLTA viscosity using K2CO3: 
	NOTE: Viscosity of PLGADA does not have to be modified using K2CO3 because the viscosity of 5 kDa PLAGDA at 1000 mg/mL is sufficient for centering the cargo.
	<ol>
		<li>Add K2CO3 (that was isolated after the PCLTA reaction) to the PCLTA at varying concentrations including 0 mg/mL, 10 mg/mL, 20 mg/mL, 40 mg/mL, and 60 mg/mL.</li>
		<li>Measure the dynamic viscosities of the solutions by changing the shear rate from 0 to 1000 1/s using rheometry.</li>
		<li>Manually inject the cargo in the middle (refer to step 4.2 to prepare the cargo mixture) of the polybubbles that were formed using the PCL/PCLTA solutions with different concentrations of K2CO3 (step 4.1.1). Determine the optimal concentration of K2CO3 by observing which solution from step 4.1.1 can result in retention of the cargo in the middle.</li>
	</ol>
	</li>
	<li>Centering of the cargo (already shown feasibility with small molecules) with CMC
	<ol>
		<li>Mix the cargo with 5% (wt/vol) CMC in a rotator overnight to increase the viscosity of the cargo.</li>
		<li>Manually inject 2 &#181;L of cargo mixture in the polybubble and proceed with UV curing at 254 nm wavelength for 60 s at 2 W/cm2.</li>
		<li>Flash freeze the polybubbles in liquid nitrogen for 30 s and lyophilize overnight at 0.010 mBar vacuum and at -85 &#176;C.</li>
		<li>Separate the polybubbles from the dried CMC using forceps and wash with DI water to remove any residual CMC.</li>
		<li>Cut the polybubble in half and image the halves using confocal microscopy to ensure that the cargo is centered (refer to step 6 for excitation and emission wavelengths used).</li>
	</ol>
	</li>
</ol>
5. Cargo Formulation
NOTE: Polybubble formulation can house various cargo types, including small molecules, proteins, and nucleic acids.
<ol>
	<li>Based on previous studies, in the case of protein cargo, use excipients including polyethylene glycol (PEG)6, polyvinylpyrrolidone (PVP), and glycopolymers6 to improve the stability of protein during polybubble formulation.</li>
	<li>Form polybubbles based on the protocol in step 2.</li>
	<li>Prepare the antigen solution by adding 17.11 g of trehalose to 625 &#181;L of HIV gp120/41 antigen.</li>
	<li>Manually inject 1 &#181;L of antigen solution in the middle of the polybubble.</li>
	<li>Open polybubbles on days 0, 7, 14, and 21, and record the fluorescence of antigen with excitation and emission wavelengths 497 nm and 520 nm, respectively.</li>
	<li>Determine the functionality of the antigen using enzyme-linked immunosorbent assay (ELISA) and use 5% nonfat milk as a blocking buffer.</li>
</ol>
6. Release of cargo
NOTE: Small molecule or antigen can be used as the cargo type
<ol>
	<li>Small molecule
	<ol>
		<li>Incubate polybubbles with centered acriflavine in 400 &#181;L of phosphate buffer saline (PBS) at 37 &#176;C, 50 &#176;C for PLGADA polybubbles and at 37 &#176;C, 50 &#176;C, 70 &#176;C for PCL/PCLTA polybubbles. 
		NOTE: The reason why we recommend testing above body temperatures is to a) simulate the temperature (50 &#176;C) at which the polybubble reaches while lasering the gold nanorods (AuNRs) within PCL and PLGA; and b) accelerate the degradation process of PCL (50 &#176;C, 70 &#176;C).</li>
		<li>At each time point, collect the supernatants and replace with 400 &#181;L of fresh PBS.</li>
		<li>Use a plate reader to quantify the fluorescence intensities in the collected supernatants. 
		NOTE: Use ex/em of 416 nm/514 nm for acriflavine.</li>
	</ol>
	</li>
	<li>Antigen
	<ol>
		<li>Incubate polybubbles with centered bovine albumin serum (BSA)-488 in 400 &#181;L of PBS at 37 &#176;C, 50 &#176;C for PLGADA polybubbles and at 37 &#176;C, 50 &#176;C for PCL/PCLTA polybubbles.</li>
		<li>At each time point, collect the supernatants and replace with 400 &#181;L fresh PBS.</li>
		<li>Use a plate reader to quantify the fluorescence intensities in the collected supernatants. Use ex/em of 497 nm/520 nm for BSA-488. 
		NOTE: Release study at 70 &#176;C for PCL/PCLTA polybubbles should not be conducted to avoid exposing the antigen to extreme temperature.</li>
	</ol>
	</li>
</ol>
7. Toxicity
<ol>
	<li>Quantifying chlorine content in polybubbles using neutron activation analysis (NAA)
	<ol>
		<li>Use polybubbles that were lyophilized for 2, 4, 6, 20, and 24 h for this study at 0.010 mBar vacuum and at -85 &#176;C.</li>
		<li>Measure 5-9 mg of polybubbles and place them on LDPE irradiation vials.</li>
		<li>Prepare 1000 g/mL of chlorine calibration solution from national institute of standards and technology (NIST)-traceable calibration solution.</li>
		<li>Use 1- megawatts Triga reactor to carry out neutron irradiations on each sample at neutron fluence rate of 9.1 &#215; 1012 /cm2&#183;s for 600 s.</li>
		<li>Transfer the polybubbles to unirradiated vials.</li>
		<li>Use HPGe detector to obtain gamma-ray spectra for 500 s after 360 s decay intervals.</li>
		<li>Use NAA software by canberra Industries to analyze the data.</li>
	</ol>
	</li>
	<li>Quantifying chlorine content released from polybubbles using NAA
	<ol>
		<li>Incubate polybubbles that were lyophilized overnight (at 0.010 mBar vacuum and at -85 &#176;C) in 400 &#181;L of PBS at 37 &#176;C.</li>
		<li>Collect the supernatants at weeks 1, 2, and 3 after incubation.</li>
		<li>Analyze the supernatants for chlorine content using NAA using the same method as described above in step 7.1.</li>
	</ol>
	</li>
</ol>
8. AuNR Synthesis by Kittler, S., et al.8
<ol>
	<li>Prepare AuNR seeding solution by mixing 250 &#181;L of 10 mM chloroauric acid (HAuCl4), 7.5 mL of 100 mM cetrimonium bromide (CTAB), and 600 &#181;L of 10 mM ice cold sodium borohydride (NaBH4).</li>
	<li>Prepare Growth solution by mixing 40 mL of 100 mM CTAB, 1.7 mL of 10 mM HAuCl4, 250 &#181;L of silver nitrate (AgNO3), and 270 &#181;L of 17.6 mg/mL ascorbic acid to a tube.</li>
	<li>Vigorously mix 420 &#181;L of seed solution with the growth solution at 1200 rpm for 1 min. Then leave the mixture undisturbed to react for 16 h.</li>
	<li>Remove the excess reagents from the mixture by centrifuging at 8000 &#215; g for 10 min and discard the supernatant.</li>
</ol>
9. Hydrophobicization of AuNRs by Soliman, M.G., et al.9
<ol>
	<li>Adjust pH of 1.5 mL of synthesized CTAB-stabilized AuNRs to 10 using 1 mM sodium hydroxide (NaOH).</li>
	<li>Stir the solution with 0.1 mL of 0.3 mM methylated PEG (mPEG) thiol at 400 rpm overnight.</li>
	<li>Mix PEGylated AuNRs with 0.4 M dodecylamine (DDA) in chloroform at 500 rpm for 4 days.</li>
	<li>Pipet out the top organic layer containing hydrophobicized AuNRs and store at 4 &#176;C until future use.</li>
</ol>
10. NIR-activation of polybubbles
<ol>
	<li>Mix the polymer (PLGADA or PCL/PCLTA) solution with hydrophobicized AuNRs in a 1:9 (vol/vol).</li>
	<li>Add photoinitiator to the polymer-AuNR mixture in a 0.005:1 (vol/vol).</li>
	<li>Form polybubbles by injecting the polymer-AuNR mixture into a 0.92 mL glass vial with 10% CMC (wt/vol) (refer to step 2).</li>
	<li>Cure the polybubbles at 254 nm wavelength for 60 s at 2 W/cm2.</li>
	<li>Flash freeze in liquid nitrogen for 30 s and lyophilize overnight at 0.010 mBar vacuum and at -85 &#176;C.</li>
	<li>Separate the dried polybubbles using forceps and wash with DI water to remove any residual CMC.</li>
	<li>Incubate the polybubbles in 400 &#181;L of PBS at 37 &#176;C.</li>
	<li>Activate the polybubbles using 801 nm NIR laser at 8A for 5 min every Monday, Wednesday, and Friday.</li>
	<li>Take forward-looking infrared (FLIR) images of the polybubble before and after laser activation to obtain temperature values.</li>
	<li>Calculate temperature differences between before and after laser activation based on the temperature values from the FLIR images.</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+polymeric+nanoparticles+based+drug+delivery+systems.">Biodegradable polymeric nanoparticles based drug delivery systems.</a> Colloids and Surfaces B: Biointerfaces. 75 (1), 1-18 (2010).</li><li>Dai, C., Wang, B., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microencapsulation+peptide+and+protein+drugs+delivery+system.">Microencapsulation peptide and protein drugs delivery system.</a> Colloids and Surfaces B: Biointerfaces. 41 (2-3), 117-120 (2005).</li><li>Souery, W. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+and+quantifying+the+stability+of+amino+acid-based+cargo+within+polymeric+delivery+systems.">Controlling and quantifying the stability of amino acid-based cargo within polymeric delivery systems.</a> Journal of Control Release. 300, 102-113 (2019).</li><li>Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+protein+formulations.">Advanced protein formulations.</a> Protein Science. 24 (7), 1031-1039 (2015).</li><li>Lee, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ultraviolet-curable,+core-shell+vaccine+formed+via+phase+separation.">An ultraviolet-curable, core-shell vaccine formed via phase separation.</a> Journal of Biomedical Materials Research Part A. , (2019).</li><li>Kittler, S., Hickey, S. 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V., Heller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsatile+and+delayed+release+of+lysozyme+from+ointment-like+poly(ortho+esters).">Pulsatile and delayed release of lysozyme from ointment-like poly(ortho esters).</a> Journal of Controlled Release. 21 (1), 191-200 (1992).</li><li>Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instability,+stabilization,+and+formulation+of+liquid+protein+pharmaceuticals.">Instability, stabilization, and formulation of liquid protein pharmaceuticals.</a> International Journal of Pharmaceutics. 185 (2), 129-188 (1999).</li><li>Wong, D. Y., Hollister, S. J., Krebsbach, P. H., Nosrat, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(epsilon-caprolactone)+and+poly+(L-lactic-co-glycolic+acid)+degradable+polymer+sponges+attenuate+astrocyte+response+and+lesion+growth+in+acute+traumatic+brain+injury.">Poly(epsilon-caprolactone) and poly (L-lactic-co-glycolic acid) degradable polymer sponges attenuate astrocyte response and lesion growth in acute traumatic brain injury.</a> Tissue Engineering. 13 (10), 2515-2523 (2007).</li><li>Davoodi, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+delivery+systems+for+programmed+and+on-demand+release.">Drug delivery systems for programmed and on-demand release.</a> Advanced Drug Delivery Reviews. 132, 104-138 (2018).</li><li>Huang, Y. C., Lei, K. F., Liaw, J. W., Tsai, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+laser+intensity+activated+plasmonic+gold+nanoparticle-generated+photothermal+effects+on+cellular+morphology+and+viability:+a+real-time,+long-term+tracking+and+monitoring+system.">The influence of laser intensity activated plasmonic gold nanoparticle-generated photothermal effects on cellular morphology and viability: a real-time, long-term tracking and monitoring system.</a> Photochemical and Photobiological Sciences. 18 (6), 1419-1429 (2019).</li><li>Link, S., Burda, C., Nikoobakht, B., El-Sayed, M. 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Authors have nothing to disclose.

Current technologies and challenges 
Emulsion-based micro- and nanoparticles have been commonly used as drug delivery carriers. Although release kinetics of the cargo from these devices have been extensively studied, controlling burst release kinetics has been a major challenge11. Cargo versatility and functionality is also limited in emulsion-based systems owing to the exposure of cargo to excess aqueous and organic solvents. Protein-based cargo are often not compatible with micro-and nanoparticles due to the possibility of cargo denaturation and aggregation12. In addition to cargo stability, cargo kinetics is especially important in the context of vaccines because of the need for booster shots leading to seroconversion. Previous efforts to address these challenges in vaccine delivery have not been sufficiently successful, as the notion of single injection vaccine systems have been around for a couple of decades and has not yet been clinically translated.
Our polybubble vaccine delivery platform can potentially overcome the challenges with increased exposure of cargo to organic solvent by minimizing the exposed cargo volume. This technology can potentially accommodate at least two cargo compartments: cargo in the shell and cargo in the center. Polybubbles with centered cargo can be used to control the burst release of the cargo while being compatible to different cargo types, including small molecules and antigen. In this study, we used polyesters with varying degradation times, PLGADA (shorter degradation time) and PCL/PCLTA (longer degradation time), as the polymer carriers and acriflavine (small molecule) as the cargo type to demonstrate delayed burst release. In the following sections we describe the crucial steps in forming polybubbles that are capable of enabling both delayed burst release and NIR activation, especially for future on-demand delivery applications.
Cargo centering within the polybubble 
Cargo centering was one of the significant challenges that was encountered during the formulation of the polybubbles. Immediately after injection, cargo would migrate to the surface and the cargo pocket would be stabilized without bursting into the aqueous 10% CMC solution. Polybubbles with such off-centered cargo can result in earlier release due to the non-uniform thickness of the polymer surrounding the cargo. Modulating the viscosity of the polymer and the cargo was thus crucial in resolving issues related to cargo centering. Viscosity of the cargo was increased by mixing the cargo solution with 5% CMC. To increase the viscosity of the polymer, molecular weight of the polymer could have been modified. However, increasing molecular weight often results in slower polymer degradation thus causing further delay in cargo release. Viscosity of the polymer was thus modified by increasing the concentration of the polymer. Higher concentration (1000 mg/mL) was sufficient to increase the viscosity of PLGADA. However, viscosity of PCL/PCLTA was not adequate to retain the cargo in the middle. Thus, K2CO3 that was isolated after the endcapping reaction of PCLTA was used to increase the viscosity of PCLTA.
Novel delayed release 
Delayed burst release was observed from the release studies conducted using the polybubbles with centered cargo. Small molecule (acriflavine) was used as centered cargo in the polybubbles to study the release profile. Unique release profiles were observed based on the polyester used due to the difference in the degradation time of the polymers. Burst release was observed earlier in PLGADA polybubbles compared to that of PCL/PCLTA polybubbles. Early cargo release was observed in PLGADA polybubbles because PLGA degrades faster compared to PCL13. Upon successful modulation of release kinetics with two types of polyesters, we further wanted to engineer the polybubble to potentially enable on-demand release of the cargo.
NIR-activation of polybubbles 
On-demand release of the cargo with respect to timing of the patients&#39; needs has been challenging to achieve using current delivery strategies14. We hypothesized that expediting the cargo release on-demand could be possible by accelerating the polymer degradation through the use of NIR- sensitive (i.e., theranostic-enabling) agents. AuNRs have been extensively studied for their ability to be activated using NIR laser that can travel few centimeters through the skin15. CTAB-stabilized AuNRs were thus prepared based on the protocol by Kittler, S, et al. and were hydrophobicized based on the methods published by Solimon, M.G., et al. Polybubbles with hydrophobicized AuNRs in the shell were then irradiated with NIR laser at desired time points for 5 min to observe temperature change. Temperatures before and after laser were measured based on the FLIR images. Cured polymer shell helped preserve the shape of AuNRs during the laser activation thus enabling multiple NIR activations of polybubbles. This is an interesting observation because in previous literature, AuNRs have often been known to lose their rod-like shape (crucial for NIR activation) due to laser activation16. The successful laser-activation of the polybubbles with AuNRs could pave the way to control on-demand release of the cargo in the next generation of polybubbles.
Significance and future applications 
The results obtained from this study thus shows that polybubbles have the potential to be used as a novel vaccine delivery platform. Preparation of polybubbles described in this paper will further enable other researchers to use polybubbles as a delivery platform for other therapeutic applications. For example, in addition to vaccine delivery, polybubbles can also be potentially used to delivery synergistic therapeutic agents with varying release kinetics. Furthermore, polybubbles are made of polyesters that are biodegradable and have been used in many FDA-approved medical devices. We further validated the safety of polybubbles by showing that the chlorine released from polybubbles are well within the safety levels recommended by the EPA17. Thus, our novel, injectable, UV-curable polybubble platform has the potential to be used as a safe and effective drug delivery platform for a variety of cargo types.
Limitations of this technology 
The polybubble platform technology can be used as a vaccine delivery platform potentially enabling controlled release. Our studies highlight the versatility of this platform capable of delivering different cargo types, including antigens and small molecules. However, one of the current limitations of this technology is that the cargo is currently being injected manually. For scaling purposes, we are currently engineering an automated platform that will enable injection (i.e., as an array) of cargo within the polybubble and will potentially help alleviate the concerns regarding translatability of this technology.

Limited immunization coverage results in the death of 3 million people specifically caused by vaccine-preventable diseases1. Inadequate storage and transportation conditions lead to wastage of functional vaccines and thus contribute to reduced global immunization. In addition, incomplete vaccination due to not adhering to the required vaccine schedules also causes limited vaccine coverage, specifically in developing countries2. Multiple visits to medical personnel are required within the recommended period for receiving booster shots, thus limiting the percentage of population with complete vaccination. Hence, there is a need for developing novel strategies for controlled vaccine delivery to circumvent these challenges.
Current efforts towards developing vaccine delivery technologies include emulsion-based polymeric systems3,4. However, cargo is often exposed to greater quantity of organic solvent that can potentially cause aggregation and denaturation, specifically in the context of protein-based cargo5,6. We have developed a novel vaccine delivery platform, &#34;polybubbles&#34;, that can potentially house multiple cargo compartments while minimizing the volume of cargo that is exposed to solvent7. For example, in our polybubble core-shell platform, one cargo pocket of diameter 0.38 mm (SEM) is injected in the center of a 1 mm polybubble. In this case, surface area of cargo exposed to organic solvent would be approximately 0.453 mm2. After considering the packing density of spheres (microparticles) within a sphere (cargo depot), the actual volume of microparticles (10 &#181;m in diameter) that could fit in the depot is 0.17 mm3. The volume of one microparticle is 5.24x10-8 mm3 and thus number of particles microparticles that can fit the depot is ~3.2x106 particles. If each microparticle has 20 cargo pockets (as a result of double-emulsion) of 0.25 &#181;m diameter, then the surface area of cargo exposed to organic solvent is 1274 mm2. Cargo depot within the polybubble thus would have ~2800-fold less surface area exposed to organic solvent compared to that of organic solvent-exposed cargo in microparticles. Our polyester-based platform can thus potentially reduce the quantity of cargo exposed to organic solvent which can otherwise cause cargo aggregation and instability.
Polybubbles are formed based on phase-separation principle where the polyester in organic phase is injected into an aqueous solution resulting in a spherical bubble. Cargo in the aqueous phase can then be injected in the center of the polybubble. Another cargo compartment can potentially be achieved within the polybubble by mixing a different cargo with the polymer shell. The polybubble at this stage will be malleable and will then be cured to result in a solid polybubble structure with cargo in the middle. Spherical polybubbles were chosen over other geometrical shapes to increase the cargo capacity within the polybubble while minimizing the overall size of the polybubble. Polybubbles with cargo in the center were chosen to demonstrate delayed burst release. Polybubbles were also incorporated with near infrared (NIR)- sensitive (i.e., theranostic-enabling) agent, namely gold nanorods (AuNR), to cause increase in temperature of the polybubbles. This effect could potentially facilitate faster degradation and could be used for controlling kinetics in future applications. In this paper, we describe our approach to form and characterize polybubbles, to achieve delayed burst release from the polybubbles, and to incorporate AuNR within the polybubbles to cause NIR-activation.

<table><tbody><tr><td>1-Step Ultra Tetramethylbezidine (TMB)-Enzyme-Linked Immunosorbent Assay (ELISA) Substrate Solution</td><td>Thermo scientific</td><td>34028</td><td></td></tr><tr><td>2-Hydroxy-2-methylpropiophenone</td><td>TCI AMERICA</td><td>H0991</td><td></td></tr><tr><td>450 nm Stop Solution for TMB Substrate</td><td>Abcam</td><td>ab17152</td><td></td></tr><tr><td>Acryloyl chloride</td><td>Sigma Aldrich</td><td>A24109-100G</td><td></td></tr><tr><td>Acriflavine</td><td>Chem-Impex International</td><td>22916</td><td></td></tr><tr><td>Anhydrous ethyl ether</td><td>Fisher Chemical</td><td>E138-500</td><td></td></tr><tr><td>Anti-HIV1 gp120 antibody conjugated to horseradish peroxidase (HRP)</td><td></td><td></td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Fisher BioReagents</td><td>BP9700100</td><td></td></tr><tr><td>BSA-CF488 dye conjugates</td><td>Invitrogen</td><td>A13100</td><td></td></tr><tr><td>Bromosalicylic acid</td><td>Acros Organics</td><td>AC162142500</td><td></td></tr><tr><td>Carboxymethylcellulose (CMC)</td><td>Millipore Sigma</td><td>80502-040</td><td></td></tr><tr><td>Centrimonium bromide (CTAB)</td><td>MP Biomedicals</td><td>ICN19400480</td><td></td></tr><tr><td>Chloroform</td><td>Fisher Chemical</td><td>C2984</td><td></td></tr><tr><td>Coating buffer</td><td>Abcam</td><td>ab210899</td><td></td></tr><tr><td>Dichloromethane (DCM)</td><td>Sigma Aldrich</td><td>270997-1L</td><td></td></tr><tr><td>Diethyl ether</td><td>Fisher Chemical</td><td>E1384</td><td></td></tr><tr><td>Dodeacyl Amine</td><td>Acros Organics</td><td>AC117665000</td><td></td></tr><tr><td>Doxorubicin hydrochloride</td><td>Fisher BioReagents</td><td>BP251610</td><td></td></tr><tr><td>L-ascorbic acid</td><td>Acros Organics</td><td>A61 100</td><td></td></tr><tr><td>Legato 100 Syringe Pump</td><td>KD Scientific</td><td>14 831 212</td><td></td></tr><tr><td>mPEG thiol</td><td>Laysan Bio</td><td>NC0702454</td><td></td></tr><tr><td>Nonfat dry milk</td><td>Andwin Scientific</td><td>NC9022655</td><td></td></tr><tr><td>Potassium carbonate</td><td>Acros Organics</td><td>AC424081000</td><td></td></tr><tr><td>Phosphate saline buffer</td><td>Fisher BioReagents</td><td>BP3991</td><td></td></tr><tr><td>(Poly(caprolactone)</td><td>Sigma Aldrich</td><td>440744-250G</td><td></td></tr><tr><td>(Poly(caprolactone) triol</td><td>Acros Organics</td><td>AC190730250</td><td></td></tr><tr><td>Poly (lactic-co-glycolic acid) diacrylate</td><td>CMTec</td><td>280050</td><td></td></tr><tr><td>Potassium carbonate</td><td>Acros Organics</td><td>AC424081000</td><td></td></tr><tr><td>Recombinant HIV1 gp120 + gp41 protein</td><td>Abcam</td><td>ab49054</td><td></td></tr><tr><td>Silver nitrate</td><td>Acros Organics</td><td>S181 25</td><td></td></tr><tr><td>Sodium borohydride</td><td>Fisher Chemical</td><td>S678 10</td><td></td></tr><tr><td>Tetrachloroauric acid</td><td>Fisher Chemical</td><td>G54 1</td><td></td></tr><tr><td>Trehalose</td><td>Acros Organics</td><td>NC9022655</td><td></td></tr><tr><td>Triethyl amine</td><td>Acros Organics</td><td>AC157910010</td><td></td></tr></tbody></table>

production of near-infrared sensitive, core-shell vaccine delivery platform

Vaccine delivery strategies that can limit the exposure of cargo to organic solvent while enabling novel release profiles are crucial for improving immunization coverage worldwide. Here, a novel injectable, ultraviolet- curable and delayed burst release- enabling vaccine delivery platform called polybubbles is introduced. Cargo was injected into polyester-based polybubbles that were formed in 10% carboxymethycellulose -based aqueous solution. This paper includes protocols to maintain spherical shape of the polybubbles and optimize cargo placement and retention to maximize the amount of cargo within the polybubbles. To ensure safety, chlorinated solvent content within the polybubbles were analyzed using neutron activation analysis. Release studies were conducted with small molecules as cargo within the polybubble to confirm delayed burst release. To further show the potential for on-demand delivery of the cargo, gold nanorods were mixed within the polymer shell to enable near-infrared laser activation.

Polybubbles were extensively characterized using SEM and NAA. Cargo was successfully centered to result in a delayed burst release. Polybubbles were also successfully laser-activated because of the presence of AuNRs within the polybubbles.
Polybubble characterization 
Polybubbles injected in an aqueous solution without CMC resulted in a flattened polybubble due to their contact with the bottom of the glass vial (Figure 1A,B). Partial flattening was observed when 5% CMC-based aqueous solution was used in place of DI water (Figure 1C). Subsequently, 10% CMC-based aqueous solution in the glass vial resulted in polybubble being suspended in the solution and thus successful maintenance of sphericity of the polybubble (Figure 1D).
Cargo centering 
Cargo injection into the polybubble in the absence of CMC resulted in leakage causing no retention of cargo within the polybubble (Figure 3). To counter this challenge, two approaches were used: 1) viscosity of PCLTA was successfully increased using K2CO3 that was isolated after endcapping PCL triol with triacrylate (Figure 2), and 2) viscosity of the cargo was successfully increased after mixing the cargo with 5% CMC (Figure 3, Figure 4). Viscosity of the PLGADA polybubbles were sufficient to facilitate centering of the cargo and thus was not modulated using K2CO3.
Antigen functionality 
HIV gp120/41 antigen was mixed with and without trehalose before injecting into the polybubble (Figure 5). Binding efficiency of antibody to the antigen (termed as functionality) with and without trehalose was observed to have no statistically significant difference.
Release studies without laser activation 
Delayed burst releases were observed in PLGADA polybubbles with acriflavine in the middle on days 19 and 5 for polybubbles incubated at 37 &#176;C (Figure 6A) and 50 &#176;C (Figure 6B), respectively. Delayed burst releases were also observed in PCL/PCLTA polybubbles with acriflavine in the middle on days 160 and 60 for polybubbles incubated at 50 &#176;C (Figure 7A) and 70 &#176;C (Figure 7B), respectively. These release studies were conducted in the absence of laser-activatable AuNRs.
In vitro laser activation of polybubbles 
Polybubbles with AuNRs in the shell were successfully laser activated multiple times in PLGADA polybubbles (Figure 8A) and PCL/PCLTA polybubbles (Figure 8B). Temperature changes from before and after laser activation were 10 &#177; 1 &#176;C and 5 &#177; 1 &#176;C in PCL/PCLTA polybubbles with higher and lower AuNR concentration in the shell, respectively. Temperature changes observed before and after laser activation were 11 &#177; 2 &#176;C and 6 &#177; 1 &#176;C in PLGADA polybubbles with higher and lower AuNR concentration in the shell, respectively.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60569/60569fig1.jpg" /> 
Figure 1: Maintaining sphericity of polybubbles. SEM images of (A) 14 kDa PCL/300 Da PCLTA flattened polybubble due to the contact of polybubble with the bottom of the glass vial; (B) 14 kDa PCL/300 Da PCLTA polybubble from the top that was not in contact with the glass bottom; (C) the PCL/PCLTA polybubbles with lesser degree of flattening when injected into a 5% CMC solution compared to DI water solution, causing the formation of hemisphere-like shape at the point of contact with the vial; (D) polybubble that did not reach the bottom of the glass vial when injected into a 10% CMC solution, allowing for the spherical shape to be maintained. All of the scale bars indicated are 500 &#181;m. This figure has been modified from Lee et al.7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60569/60569fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60569/60569fig2.jpg" /> 
Figure 2: Modulation of PCLTA viscosity. Concentration of K2CO3 was increased from 0 to 80 mg/mL in PCLTA and dynamic viscosity was observed to increase proportionally with the concentration of K2CO3.&#160;This figure has been modified from Arun Kumar et al10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60569/60569fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60569/60569fig3.jpg" /> 
Figure 3: Cargo injection into the polybubble with and without CMC. Top panel shows frames extracted from the video of cargo leakage during injection in the absence of CMC. Bottom panel shows frames extracted from the video of cargo retention within the polybubble in the presence of 5% CMC. This figure has been modified from Lee et al.7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60569/60569fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60569/60569fig4.jpg" /> 
Figure 4: Centered cargo. Fluorescent microscope images of (A) PCL/PCLTA polybubble with centered cargo, (B) PCL/PCLTA polybubble with cargo in the shell and centered non-fluorescent dye.&#160;This figure has been modified from Arun Kumar et al10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60569/60569fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60569/60569fig5.jpg" /> 
Figure 5: Antigen functionality with trehalose. Functionality of HIV gp120/41 with and without trehalose within the polybubble was analyzed using ELISA. The binding efficiency of an antibody to the protein is generally regarded as an indicator for the functionality of the protein. When we discuss the functionality of antigen in this study, we intend it to mean that it aids the antibodies binding the protein of interest (which is an indicator for protein functionality). No statistical significance was observed between the two groups. Confidence intervals are indicated by solid and dotted lines. This figure has been modified from Lee et al.7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60569/60569fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60569/60569fig6.jpg" /> 
Figure 6: Delayed burst release from PLGADA polybubbles. Release studies showing delayed burst releases from PLGADA polybubbles with acriflavine in the middle at (A) 37 &#176;C, (B) 50 &#176;C. Solid line indicates the fitted curve obtained based on the data points.&#160;This figure has been modified from Arun Kumar et al10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60569/60569fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60569/60569fig7.jpg" /> 
Figure 7: Delayed burst release from PCL/PCLTA polybubbles. Release studies showing delayed burst releases from PCL/PCLTA polybubbles with acriflavine in the middle at (A) 50 &#176;C, (B) 70 &#176;C.&#160;This figure has been modified from Arun Kumar et al10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60569/60569fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/60569/60569fig8.jpg" /> 
Figure 8: NIR laser activation of polybubbles. Temperature change observed before and after NIR laser activation in (A) PLGADA polybubbles, (B) PCL/PCLTA polybubbles with higher and lower concentration of AuNRs in the polymer shell. This increase in temperature could be leveraged to potentially expedite the polymer degradation leading to earlier release of the cargo.&#160;This figure has been modified from Arun Kumar et al10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60569/60569fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Production of Near-Infrared Sensitive, Core-Shell Vaccine Delivery Platform at JoVE.com

Production of Near-Infrared Sensitive, Core-Shell Vaccine Delivery Platform

This article describes the protocols used to produce a novel vaccine delivery platform, &#34;polybubbles,&#34; to enable delayed burst release. Polyesters including poly(lactic-co-glycolic acid) and polycaprolactone were used to form the polybubbles and small molecules and antigen were used as cargo.

Vaccine delivery strategies that can limit the exposure of cargo to organic solvent while enabling novel release profiles are crucial for improving ...

production-near-infrared-sensitive-core-shell-vaccine-delivery

Nanyang Technological University

Research

JoVE Journal

Biology

5.1K Views.  Texas A&M University. This article describes the protocols used to produce a novel vaccine delivery platform, "polybubbles," to enable delayed burst release. Polyesters including poly(lactic-co-glycolic acid) and polycaprolactone were used to form the polybubbles and small molecules and antigen were used as cargo.

Video: Production of Near-Infrared Sensitive, Core-Shell Vaccine Delivery Platform

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Immunology and Infection

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different infectious diseases. In this article we demonstrate a method to produce a SAPN core containing a six-helix bundle (SHB) assembly that is capable of presenting antigens in a trimeric conformation. We describe the expression of the SHB-SAPN in an E. coli system, as well as the necessary protein purification steps. We included an isopropanol wash step to reduce the residual bacterial lipopolysaccharide. As an indication of the protein identity and purity, the protein reacted with known monoclonal antibodies in Western blot analyses. After refolding, the size of the particles fell in the expected range (20 to 100 nm), which was confirmed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. The methodology described here is optimized for the SHB-SAPN, however, with only slight modifications it can be applied to other SAPN constructs. This method is also easily transferable to large scale production for GMP manufacturing for human vaccines.

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...

Bioengineering

A "Plug-And-Display" Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain

Biomimetic nanoparticles obtained from bacteria or viruses have attracted substantial interest in vaccine research and development. Outer membrane vesicles (OMVs) are mainly secreted by gram-negative bacteria during average growth, with a nano-sized diameter and self-adjuvant activity, which may be ideal for vaccine delivery. OMVs have functioned as a multifaceted delivery system for proteins, nucleic acids, and small molecules. To take full advantage of the biological characteristics of OMVs, bioengineered Escherichia coli-derived&#160;OMVs were utilized as a carrier and SARS-CoV-2 receptor-binding domain (RBD) as an antigen to construct a &#34;Plug-and-Display&#34; vaccine platform. The SpyCatcher (SC) and SpyTag (ST) domains in Streptococcus pyogenes were applied to conjugate OMVs and RBD. The Cytolysin A (ClyA) gene was translated with the SC gene as a fusion protein after plasmid transfection, leaving a reactive site on the surface of the OMVs. After mixing RBD-ST in a conventional buffer system overnight, covalent binding was formed between the OMVs and RBD. Thus, a multivalent-displaying OMV vaccine was achieved. By replacing with diverse antigens, the OMVs vaccine platform can efficiently display a variety of heterogeneous antigens, thereby potentially rapidly preventing infectious disease epidemics. This protocol describes a precise method for constructing the OMV vaccine platform, including production, purification, bioconjugation, and characterization.

The present protocol describes the bioengineering of outer membrane vesicles to be a "Plug-and-Display" vaccine platform, including production, purification, bioconjugation, and characterization.

Biomimetic nanoparticles obtained from bacteria or viruses have attracted substantial interest in vaccine research and development. Outer membrane ...

Cellular Affinity of Particle-Stabilized Emulsion to Boost Antigen Internalization

The cellular affinity of micro-/nanoparticles is the precondition for cellular recognition, cellular uptake, and activation, which are essential for drug delivery and immune response. The present study stemmed from the observation that the effects of charge, size, and shape of solid particles on cell affinity are usually considered, but we seldom realize the essential role of softness, dynamic restructuring phenomenon, and complex interface interaction in cellular affinity. Here, we developed poly-lactic-co-glycolic acid (PLGA) nanoparticle-stabilized Pickering emulsion (PNPE) that overcame the shortcomings of rigid forms and simulated the flexibility and fluidity of pathogens. A method was set up to test the affinity of PNPE to cell surfaces and elaborate on the subsequent internalization by immune cells. The affinity of PNPE to bio-mimetic extracellular vesicles (bEVs)-the replacement for bone marrow dendritic cells (BMDCs)-was determined using a quartz crystal microbalance with dissipation monitoring (QCM-D), which allowed real-time monitoring of cell-emulsion adhesion. Subsequently, the PNPE was used to deliver the antigen (ovalbumin, OVA) and the uptake of the antigens by BMDCs was observed using confocal laser scanning microscope (CLSM). Representative results showed that the PNPE immediately decreased frequency (&#916;F) when it encountered the bEVs, indicating rapid adhesion and high affinity of the PNPE to the BMDCs. PNPE showed significantly stronger binding to the cell membrane than PLGA microparticles (PMPs) and AddaVax adjuvant (denoted as surfactant-stabilized nano-emulsion [SSE]). Furthermore, owing to the enhanced cellular affinity to the immunocytes through dynamic curvature changes and lateral diffusions, antigen uptake was subsequently boosted compared with PMPs and SSE. This protocol provides insights for designing novel formulations with high cell affinity and efficient antigen internalization, providing a platform for the development of efficient vaccines.

To rationally design efficient adjuvants, we developed poly-lactic-co-glycolic acid nanoparticle-stabilized Pickering emulsion (PNPE). The PNPE possessed unique softness and a hydrophobic interface for potent cellular contact and offered high-content antigen loading, improving the cellular affinity of the delivery system to antigen-presenting cells and inducing efficient internalization of antigens.

The cellular affinity of micro-/nanoparticles is the precondition for cellular recognition, cellular uptake, and activation, which are essential for drug ...

Fabrication of Pulsatile Polymeric Microparticles Encapsulating Rabies Antigen

The current guidelines for rabies post-exposure prophylaxis require multiple injections administered over several weeks. This can be disproportionately burdensome to those living in low- and middle-income countries (LMICs), where the majority of deadly exposures to rabies occur. Different drug delivery strategies have been explored to condense vaccine regimens to a single injection by encapsulating antigens into polymeric particles. However, harsh stressors during the encapsulation process can cause denaturation of the encapsulated antigen. This article describes a method for encapsulating the rabies virus (RABV) antigen into polymeric microparticles that exhibit tunable pulsatile release. This method, termed Particles Uniformly Liquified and Sealed to Encapsulate Drugs (PULSED), generates microparticles using soft lithography to create inverse polydimethylsiloxane (PDMS) molds from a multi-photon, 3D-printed master mold. Poly(lactic-co-glycolic acid) (PLGA) films are then compression-molded into the PDMS molds to generate open-faced cylinders that are filled with concentrated RABV using a piezoelectric dispensing robot. These microstructures are then sealed by heating the top of the particles, allowing the material to flow and form a continuous, nonporous polymeric barrier. Post-fabrication, an enzyme-linked immunosorbent assay (ELISA) specific to the detection of intact trimeric rabies virus glycoprotein is used to confirm the high recovery of immunogenic antigen from the microparticles.

This method describes the encapsulation of the rabies antigen into biodegradable polymeric microparticles with structural and material properties that enable pulsatile release after a predetermined delay. Enzyme-linked immunosorbent assay (ELISA) assessment of the antigen retrieved from the particle core confirms the presence of intact trimeric rabies virus glycoprotein through particle fabrication.

The current guidelines for rabies post-exposure prophylaxis require multiple injections administered over several weeks. This can be disproportionately ...

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard manufacturing. To achieve an effective protein-based subunit vaccine, the protein antigen often needs to adopt a native-like conformation. This is particularly important for pathogen-surface antigens that are membrane-bound proteins. Cell-free methods have been successfully used to produce correctly folded functional membrane protein through the co-translation of nanolipoprotein particles (NLPs), commonly known as nanodiscs. 
This strategy can be used to produce subunit vaccines consisting of membrane proteins in a lipid-bound environment. However, cell-free protein production is often limited to small scale (&lt;1 mL). The amount of protein produced in small-scale production runs is usually sufficient for biochemical and biophysical studies. However, the cell-free process needs to be scaled up, optimized, and carefully tested to obtain enough protein for vaccine studies in animal models. Other processes involved in vaccine production, such as purification, adjuvant addition, and lyophilization, need to be optimized in parallel. This paper reports the development of a scaled-up protocol to express, purify, and formulate a membrane-bound protein subunit vaccine. 
Scaled-up cell-free reactions require optimization of plasmid concentrations and ratios when using multiple plasmid expression vectors, lipid selection, and adjuvant addition for high-level production of formulated nanolipoprotein particles. The method is demonstrated here with the expression of a chlamydial major outer membrane protein (MOMP) but may be widely applied to other membrane protein antigens. Antigen effectiveness can be evaluated in vivo through immunization studies to measure antibody production, as demonstrated here.

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

This protocol describes using commercial, cell-free protein expression kits to produce membrane proteins supported in nanodisc that can be used as antigens in subunit vaccines.

Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard ...

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; in particular, some MHC I-restricted epitopes can induce effective cytotoxic T lymphocyte activity to clear tumor cells. Additionally, nasal administration is an effective and safe delivery technique for tumor vaccines due to its convenience and improved patient compliance. However, epitope peptides are unsuitable for nasal delivery because of their poor immunogenicity and lack of delivery efficiency. Nanoemulsions (NEs) are thermodynamically stable systems that can be loaded with antigens and delivered directly to the nasal mucosal surface. Ile-Lys-Val-Ala-Val (IKVAV) is the core pentapeptide of laminin, an integrin-binding peptide expressed by human respiratory epithelial cells. In this study, an intranasal self-assembled epitope peptide NE tumor vaccine containing the synthetic peptide IKVAV-OVA257-264 (I-OVA) was prepared by a low-energy emulsification method. The combination of IKVAV and OVA257-264 can enhance antigen uptake by nasal mucosal epithelial cells. Here, we establish a protocol to study the physicochemical characteristics by transmission electron microscopy (TEM), atomic force microscopy (AFM), and dynamic light scattering (DLS); stability in the presence of mucin protein; toxicity by examining the cell viability of BEAS-2B cells and the nasal and lung tissues of C57BL/6 mice; cellular uptake by confocal laser scanning microscopy (CLSM); release profiles by imaging small animals in vivo; and the protective and therapeutic effect of the vaccine by using an E.G7 tumor-bearing model. We anticipate that the protocol will provide technical and theoretical clues for the future development of novel T cell epitope peptide mucosal vaccines.

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Here, we present detailed methods for the preparation and evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in vivo.

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; ...

Evaluating Immunogenicity of Nanoemulsion&#45;Encapsulated Retinoic Acid in Mice

Cationic Nanoemulsion-Encapsulated Retinoic Acid as an Adjuvant to Promote OVA&#45;Specific Systemic and Mucosal Responses

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen uptake and then promotes antigen-specific immune responses. In recent years, retinoic acid (RA) has received increasing attention due to its effect in activating the mucosal immune response; however, in order to use RA as a mucosal adjuvant, it is necessary to solve the problem of its dissolution, loading, and delivery. Here, we describe a cationic nanoemulsion-encapsulated retinoic acid (CNE-RA) delivery system composed of the cationic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOTAP), retinoic acid, squalene as the oil phase, polysorbate 80 as surfactant, and sorbitan trioleate 85 as co-surfactant. Its physical and chemical properties were characterized using dynamic light scattering and a spectrophotometer. Immunization of mice with the mixture of antigen (ovalbumin, OVA) and CNE-RA significantly elevated the levels of anti-OVA secretory immunoglobulin A (sIgA) in vaginal lavage fluid and the small intestinal lavage fluid of mice compared with OVA alone. This protocol describes a detailed method for the preparation, characterization, and evaluation of the adjuvant effect of CNE-RA.

Author Spotlight: Development and Evaluation of a Cationic Nanoemulsion&#45;Encapsulated Retinoic Acid System for Mucosal Vaccination

In this protocol, we developed a cationic nanoemulsion-encapsulated retinoic acid (RA) to be used as an adjuvant to promote antigen-specific systemic and mucosal responses. By adding the FDA-approved RA to the nanoemulsion, antigen-specific sIgA was promoted in the vagina and small intestine after intramuscular injection of the nanoemulsion.

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen ...

Generating a Multivalent-Displaying Outer Membrane Vesicle Vaccine

Source: Feng, R., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64213">A &#34;Plug-And-Display&#34; Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain.</a> J. Vis. Exp. (2022).
This video demonstrates a method to generate multivalent-displaying outer membrane vesicle (OMV) vaccines. Mammalian cells are transfected to express and secrete viral antigens fused to a SpyTag and a polyhistidine tag. These antigens are then purified using a nickel-nitrilotriacetic acid (Ni-NTA) affinity column. The purified antigen is mixed with outer membrane vesicles expressing surface-bound SpyCatcher (SC) protein. Multiple antigens covalently attach to SpyCatchers via their SpyTags (ST), creating OMVs that display the antigen, serving as a multivalent vaccine.

Wytwarzanie wielowalentnej szczepionki pęcherzykowej z błoną zewnętrzną

1. Plasmid construction

	Insert DNA encoding SpyCatcher sequence (Figure 1) into an ampicillin-resistant pThioHisA-Cytolysin A (ClyA) plasmid (see Table ...

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Immunogenics and Vaccine Development

A Technique to Generate a Novel Delivery Platform for a Delayed Burst Release of Molecules

Source: Arun Kumar, S. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60569">Production of Near-Infrared Sensitive, Core-Shell Vaccine Delivery Platform.</a>&#160;J. Vis. Exp. (2020)
This video demonstrates a method for the generation of a polymer bubble or polybubble-based novel platform that enables delayed burst release. Polyester-based polymers are used to form the polybubbles with core-shell structure, and small molecules and antigens can be used as cargo.

Technika generowania nowatorskiej platformy dostarczania do opóźnionego uwalniania cząsteczek w trybie burst

1. Polycaprolactone triacrylate (PCLTA) synthesis

	Dry 3.2 mL of 400 Da polycaprolactone (PCL) triol overnight at 50 &#176;C in an open 200 mL round ...

Production of Near-Infrared Sensitive, Core-Shell Vaccine Delivery Platform

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Cationic Nanoemulsion-Encapsulated Retinoic Acid as an Adjuvant to Promote OVA-Specific Systemic and Mucosal Responses

Wytwarzanie wielowalentnej szczepionki pęcherzykowej z błoną zewnętrzną

Technika generowania nowatorskiej platformy dostarczania do opóźnionego uwalniania cząsteczek w trybie burst